A complete understanding of the biochemical pathways used for insulin signalling is essential for understanding pathogenesis of diabetes mellitus and designing new therapies. We identified a serine/threonine kinase, termed MAP kinase, which functions in an insulin-stimulated protein kinase cascade to mediate at least some of the cellular actions of insulin, including its hallmark-activation of glycogen synthase. To define the upstream pathways for activation of MAP kinase, we are focusing on the following questions, utilizing both biochemical and molecular genetic approaches. 1. Characterization of cDNAs for MAP Kinase Kinase (MAPKK). We have isolated and sequenced a cDNA (K28) that encodes a MAPKK. In addition, the strategy for molecular cloning allowed isolation of additional clones at least one of which (K5) encodes an additional MAPKK related protein. Analysis of the clones will be completed and all distinct MAPKK homologs therein characterized. 2. Utilization of MAPKK cDNAs to generate reagents needed for mechanistic studies. Development of several reagents and procedures are necessary preliminaries for critical biochemical investigations of the upstream pathways: recombinant MAPKK(s), anti-MAPKK antibodies, site-directed mutants of MAPKKs. 3. Analysis of Mechanism(s) of activation of MAPKK by insulin. Characterization of insulin-stimulated phosphorylation of MAPKK in intact cells. Determination of the basis of MAPKK protein heterogeneity. Identification and purification of insulin-stimulated activator(s) of MAPKK by reactivation assays. Assessment of nuclear targeting of MAPKK. 4. Investigation of the role of p21 ras in activation of MAPKK by insulin. Several studies point to p21 ras as an upstream component of the MAP kinase pathway and will be pursued by: development of an in vitro system for activation of MAP kinase by p21 ras in mammalian cells and utilization of the system to identify and purify the target protein for p21 ras responsible for activation of MAPKK. 5. Examination of possible association/interaction of upstream components of MAP kinase pathway with Insulin Receptor Substrate 1 (IRS-1) (collaborative with Morris White, Joslin Diabetes Center) Tyrosine phosphorylated IRS-1 is hypothesized to be a 'docking' protein for signal transducing proteins, and therefore selective examination of associations of IRS-1 with components of the MAP kinase pathway will be made.